CN107119118B - Application of BANCR long-chain non-coding RNA and small-molecule inhibitor thereof in inhibiting ovarian cancer liver metastasis - Google Patents
Application of BANCR long-chain non-coding RNA and small-molecule inhibitor thereof in inhibiting ovarian cancer liver metastasis Download PDFInfo
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Abstract
The invention discloses application of BANCR long-chain non-coding RNA and a small molecule inhibitor thereof in inhibiting ovarian cancer liver metastasis. The invention discovers that the pyrrolopyrimidinone I, the pyrrolopyrimidinone II and the pyrrolopyrimidinone III can obviously inhibit the expression of LncRNA BANCR in HO-8910PM cells, thereby obviously inhibiting the motor invasion capacity of the HO-8910PM cells and inhibiting the liver invasion and liver metastatic cancer formation capacity of the HO-8910PM cells in nude mice. The inhibitor of the pyrrolopyrimidinone I, the pyrrolopyrimidinone II, the pyrrolopyrimidinone III and other LncRNA BANCR can be used for developing and preparing a medicament for inhibiting ovarian cancer liver metastasis. The invention has outstanding substantive features and obvious progress.
Description
Technical Field
The invention belongs to the field of gene therapy, relates to an application of a gene target and an inhibitor thereof, and particularly relates to an application of an LncRNABANCR and a small molecular inhibitor thereof in inhibiting ovarian cancer liver metastasis.
Background
The liver is one of the organs that frequently develop malignant tumor metastasis. Surgical resection is an effective treatment for metastatic cancer of the liver. However, about 80-90% of patients with liver metastatic cancer cannot be surgically resected due to uncompensated liver function, large and numerous metastatic cancers, progression of primary disease, and the like. For liver metastatic cancer which can not be resected by operation, systemic chemotherapy, interventional therapy through hepatic artery or palliative therapy are mostly adopted, and the effect is not satisfactory. Inhibition of hepatic metastasis potential of malignant tumors at the molecular level by gene therapy is a desire to reduce incidence of hepatic metastasis of malignant tumors and improve prognosis (expanding genetic x-expression signatures for predicting metabolic free area after reaction of metabolic cancer promoters, PLoS One, 2012; Inhibition of chemokinetic expression on genetic tumor by CXCR4 and its effects on genetic tumor promoters, invest optoholmol vis Sci, 2009).
Non-coding RNA refers to RNA that does not code for a protein, including rRNA, tRNA, snRNA, snorRNA, miRNA, and long non-coding RNA (Lnc RNA). Lnc RNA is a class of RNA molecules with a length of more than 200 bases, and due to lack of an effective open reading frame, it does not encode protein, but has complex biological functions and plays an important role in various biological processes, such as chromatin modification, inactivation of X chromosome, involvement in gene transcription, translation, and regulation of protein activity, etc., and its variation and regulation can cause multiple diseases including tumor. The abnormally expressed Lnc RNA can participate in various stages of tumor occurrence, development, invasion and metastasis through different pathways and different action mechanisms, and is a key factor of tumor progression. In recent years, researches show that the abnormally expressed Lnc RNA participates in the regulation and control of apoptosis, proliferation, invasion, metastasis and the like of tumor cells through multiple ways, and has close relation with the occurrence and metastasis of tumors such as liver cancer and the like.
BRAF activated non-coding RNA (BANCR) is 693bp long-chain non-coding RNA, is firstly found in melanoma cells, and LncRNA BANCR expression is abnormally found in papillary thyroid cancer, retinoblastoma, lung cancer, gastric cancer, colorectal cancer and liver cancer.
At present, no research has proved that LncRNA BANCR and small molecule inhibitors thereof are related to the inhibition of ovarian cancer liver metastasis.
Disclosure of Invention
The invention aims to provide an LncRNA BANCR and an application of a small molecule inhibitor thereof in inhibiting ovarian cancer liver metastasis.
The technical scheme for realizing the aim of the invention is as follows:
an application of LncRNA BANCR as a drug target in the preparation of drugs for inhibiting ovarian cancer liver metastasis.
Application of LncRNA BANCR inhibitor in preparing medicine for inhibiting ovarian cancer liver metastasis.
Preferably, the inhibitor is a pyrrolopyrimidinone small molecule compound with the following structural general formula,
wherein R is alkyl.
Preferably, the inhibitor is selected from compounds of the following structure:
a pharmaceutical composition for inhibiting liver metastasis of ovarian cancer comprises any one of the above inhibitors.
The invention has the outstanding advantages that:
the invention discovers that the pyrrolopyrimidinone I, the pyrrolopyrimidinone II and the pyrrolopyrimidinone III can obviously inhibit the expression of LncRNA BANCR in HO-8910PM cells, thereby inhibiting the motor invasion of the HO-8910PM cells and inhibiting the ability of the HO-8910PM cells to invade the liver in nude mice to form liver metastatic cancer. The inhibitor of the pyrrolopyrimidinone I, the pyrrolopyrimidinone II, the pyrrolopyrimidinone III and other LncRNA BANCR can be used for developing and preparing a medicament for inhibiting ovarian cancer liver metastasis.
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FIG. 1 is the relative expression level of BANCR mRNA in various groups of HO-8910PM cells;
FIG. 2 is a graph showing the relative expression levels of E-cadherin and Vimentin in the HO-8910PM cells of each group;
FIG. 3 shows the inhibition (%) of cell motility and inhibition (%) of invasion for each group of HO-8910 PM;
FIG. 4 shows the incidence (%) of metastatic liver cancer in each group.
Detailed Description
The following detailed description of the present invention is provided in connection with the examples, and for reasons of brevity, the description of the experimental procedures is not intended to be exhaustive, and all parts not specifically described in the experiments are routine procedures well known to those skilled in the art.
First, experimental material
Human ovarian cancer highly metastatic cells HO-8910PM was purchased from Sainbur (Shanghai) Biotechnology GmbH. Healthy female BALB/c nude mice of 4 weeks of age, weighing 19-21g, purchased from Nanjing university animal center.
Pyrrolopyrimidinones I, II and III were synthesized by the company's department of synthesis according to the literature methods and the structures were confirmed by nuclear magnetism. Dissolved in DMSO to prepare a 1mg/mL stock solution, and diluted to different concentrations as required. RPMI-1640 medium was purchased from Gibco; fetal calf serum was purchased from Hangzhou ilex bioengineering and materials research institute; transwell chambers were purchased from Corning corporation.
Second, Experimental methods
1. Cell culture and grouping
And (5) recovering and culturing human ovarian cancer cells HO-8910 PM. Culturing in RPMI-1640 medium (containing double antibody) containing 10% fetal calf serum at 37 deg.C under 5% CO2And a thermostated incubator with a relative humidity of 95%. After digestion and passage, the cells in logarithmic growth phase are cultured and tested. Trypan blue dye exclusion was used before the experiment to ensure that the dye exclusion rate of the cells used was above 95%.
The group of administration components and the administration concentrations were as follows:
pyrrolopyrimidinones group i: the culture solution contains pyrrolopyrimidinone I with final concentration of 5 mu M and 20 mu M;
pyrrolopyrimidinone group ii: the culture solution contains pyrrolopyrimidinone II with final concentration of 5 mu M and 20 mu M;
pyrrolopyrimidinones group iii: the culture medium contained pyrrolopyrimidinone III at a final concentration of 5. mu.M and 20. mu.M.
2. RNA extraction and RT-PCR detection of BANCR mRNA relative expression level
After HO-8910PM cells are digested and counted, the cells are added into a 6-well plate, and the cell density is 2 × 105and/mL, after the cells are attached to the wall, 5 mu M and 20 mu M of pyrrolopyrimidinone are added for culturing for 24h, the cells are collected, and the relative expression level of BANCR is determined. A control group was also set, and no pyrrolopyrimidinone was added to the control group. Total RNA was extracted using TRIzol reagent (Invitrogen). Total RNA (500ng) was reverse transcribed to a final volume of 10. mu.L using random primers from PrimeScript RT kit (TaKaRa) under standard conditions. The BANCR expression level was measured according to the instructions of STBR Premix Ex Taq (TaKaRa). Through 2-ΔΔCtMethod, the relative expression level of BANCR mRNA is calculated according to the content of internal reference GAPDH.
RT-PCR primers for LncRNAPANCR and GAPDH were as follows:
BANCR upstream primer: 5'-ACAGGACTCCATGGCAAACG-3', respectively;
BANCR downstream primer: 5'-ATGAAGAAAGCCTGGTGCAGT-3', respectively;
GAPDH upstream primer: 5'-ACCACAGTCCATGCCATCAC-3', respectively;
GAPDH downstream primer: 5'-TCCACCACCCTGTTGCTGTA-3' are provided.
3. Western blot detection of protein expression
The expression level of BANCR can be indirectly obtained by detecting the concentration of E-cadherin and Vimentin proteins.
After HO-8910PM cells are digested and counted, the cells are added into a 6-well plate, and the cell density is 2 × 105and/mL, after the cells are attached to the wall, adding 5 mu M and 20 mu M of pyrrolopyrimidinone, culturing for 24h, collecting the cells, and determining the relative expression levels of the E-cadherin protein and the Vimentin protein. A control group was also set, and no pyrrolopyrimidinone was added to the control group. The mammalian protein extraction reagent RIPA was used and supplemented with a portion of the protease inhibitor cocktail and phenylmethylsulfonyl fluoride to lyse cells. Protein concentrations were determined using a Bio-Rad protein assay kit. A10% SDS-PAGE experiment was performed on 50. mu.g of the protein extract, and the protein was transferred to nitrocellulose membrane (Sigma) and purified by 1: 1000 dilution of E-cadherin antibody (BD Co.), Vimentin antibody (Cell Signaling technology Co.). Autoradiographs were quantified by a gel image processing system (QuantityOne software, Bio-Rad) and the relative expression levels of E-cadherin and Vimentin proteins were determined using GAPDH as a control.
4. Transwell method for detecting HO-8910PM cell movement invasion capacity
600. mu.L of RPMI-1640 medium containing 10. mu.g/mL fibronectin per well was added to a 24-well plate, and HO-8910PM cells in the logarithmic growth phase were collected, resuspended in RPMI-1640 medium supplemented with pyrrolopyrimidinone (5. mu.M, 20. mu.M) and without pyrrolopyrimidinone, respectively, and adjusted to a concentration of 1 × 106and/mL. Transwell chambers were immersed in conditioned medium in 24-well plates, 100. mu.L of cell suspension was added to each chamber, and the cells were incubated at 37 ℃ in 5% CO2Culturing for 6h in an incubator. The Transwell chamber was removed, the filter membrane was fixed with methanol for 1min, stained with crystal violet for 15min, washed with water, cotton swab carefully wiped of non-transmembrane cells, gel-loaded with neutral gum, and the number of fine membrane-penetrating cells counted under 400-fold microscopeThe number of cells. Every membrane counts 5 random different visual fields in the upper, lower, left and right, and each group is provided with 3 filter membranes in parallel. The exercise inhibition (%) was calculated according to the following formula. In the invasion test, 5. mu.g (10. mu.L) of basement membrane ingredient Matrigel was coated on the inner surface of the membrane of the Transwell chamber, and the membrane was dried by air overnight on an ultra-clean bench to form an artificially reconstituted basement membrane, and the rest of the procedure was the same as that of the cell movement test. The invasion inhibition (%) was calculated according to the following formula:
the rate of exercise inhibition (%) (number of control transmembrane cells-number of administration group transmembrane cells)/number of control transmembrane cells × 100%;
the invasion inhibition ratio (%) (number of control transmembrane cells-number of administration group transmembrane cells)/number of control transmembrane cells × 100%.
5. Cancer cell metastasis capability observation of nude mouse ovarian cancer liver metastasis model
40 nude mice are raised in SPF environment, randomly divided into pyrrolopyrimidinone I group, pyrrolopyrimidinone II group, pyrrolopyrimidinone III group and normal control group, 10 mice in each group are subjected to ovarian cancer cell tumorigenic experiment, the nude mice are weighed, anesthetized, opened, spleen is exposed, HO-8910PM cells in logarithmic growth phase 1 × 10 are collected6After being resuspended in 0.1ml of physiological saline, the suspension was injected slowly into the spleen of a nude mouse for about 5 min. Hemostasis is performed for 3min by pressing the injection needle hole, and the abdomen is closed after tumor is transferred through the abdominal cavity, and the operation process follows the principle of no tumor. The cancer cells of nude mice are planted on the same day, 5mg/kg of pyrrolopyrimidinone I, pyrrolopyrimidinone II and pyrrolopyrimidinone III are respectively injected into tail veins of each group every day, and the same amount of physiological saline is injected into a control group and is continuously injected for 28 days. And (3) taking the liver of the nude mouse 8 hours after the last injection, continuously slicing the liver with the thickness of 2mm, observing whether liver metastatic cancer is formed, counting the number of nude mice with liver metastatic cancer and the average tumor forming number, and calculating the incidence (%) of the liver metastatic cancer according to the following formula:
the incidence (%) of liver metastasis cancer is the number of nude mice with liver metastasis/total number of nude mice in this group × 100%.
In the experimental process, no nude mice in each group die, and the total number of the nude mice in each group is 10.
6. Statistical method
The data analysis was performed using SPSS16.0 statistical software, with P <0.05 as the difference being statistically significant.
Third, experimental results
1. Effect of pyrrolopyrimidinones on the relative expression levels of BANCR mRNA in HO-8910PM cells
Compared with a control group, the relative expression level of BANCR mRNA of HO-8910PM cells of the administration groups of pyrrolopyrimidinone I, pyrrolopyrimidinone II and pyrrolopyrimidinone III is remarkably reduced (P is less than 0.05), and the relative expression level of BANCR mRNA of HO-8910PM cells of a high concentration group (20 mu M) is more remarkably reduced than that of a low concentration group (5 mu M). Table 1 and FIG. 1 show the comparison of the relative expression levels of BANCR mRNA from various groups of HO-8910PM cells.
TABLE 1 relative BANCR mRNA expression levels of the groups HO-8910PM cells
2. Effect of pyrrolopyrimidinones on BANCR expression levels in HO-8910PM cells
LncRNA BANCR is known to be involved in the regulation of endothelial cell keratinization pathway, the expression of LncRNA BANCR is down-regulated to result in vismentin down-regulation and E-cadherin up-regulation, and the expression level of BANCR can be indirectly obtained by detecting the concentrations of E-cadherin and vismentin protein. Compared with a control group, the expression level of the pyrrolopyrimidinone I, pyrrolopyrimidinone II and pyrrolopyrimidinone III administration group HO-8910PM cells Vimentin is remarkably reduced (P < 0.05), the expression level of E-cadherin is remarkably increased (P < 0.05), and the reduction or increase of the high concentration group (20 mu M) is more remarkable than that of the low concentration group (5 mu M). Table 2 and FIG. 2 show the comparison of the relative expression levels of E-cadherin and Vimentin in the individual groups of HO-8910PM cells.
TABLE 2 relative expression levels of E-cadherin and Vimentin from each group of HO-8910PM cells
3. Effect of pyrrolopyrimidinones on HO-8910PM cell locomotor invasion Capacity
Compared with the control group, the motility and invasion capacity of the cells of the pyrrolopyrimidinone I, pyrrolopyrimidinone II and pyrrolopyrimidinone III administration group HO-8910PM are remarkably reduced (P is less than 0.05), and the inhibition effect of the high concentration group (20 mu M) is more remarkable than that of the low concentration group (5 mu M). Table 3 and FIG. 3 show the inhibition ratios (%) of cell movement and the inhibition ratios (%) of invasion for each group of HO-8910 PM.
Table 3 inhibition (%) of cell movement and inhibition (%) of invasion for each group HO-8910PM (%)
4. Effect of pyrrolopyrimidinones on incidence of liver metastasis of ovarian cancer in nude mice
During the observation period, no nude mice died in each group, and the total number of the nude mice in each group was 10. Compared with a control group, the incidence rate (%) of liver metastatic cancers of the pyrrolopyrimidinone I, pyrrolopyrimidinone II and pyrrolopyrimidinone III administration groups is remarkably reduced (P < 0.05).
The number of nude mice with liver metastasis and the incidence (%) of liver metastatic cancer in each group are shown in Table 4 and FIG. 4.
Table 4 number of nude mice with liver metastasis and incidence of liver metastatic cancer (%)
| Number of nude mice (only) with liver metastasis | Incidence of liver metastatic cancer (%) | |
| Pyrrolopyrimidinones group I | 1 | 10 |
| Pyrrolopyrimidinones group II | 1 | 10 |
| |
1 | 10 |
| |
10 | 100 |
The experiment can find that the pyrrolopyrimidinone I, the pyrrolopyrimidinone II and the pyrrolopyrimidinone III can obviously inhibit the expression of LncRNA BANCR in HO-8910PM cells, thereby obviously inhibiting the motor invasion capacity of the HO-8910PM cells and inhibiting the liver invasion and liver metastatic cancer formation capacity of the HO-8910PM cells in nude mice. The inhibitor of the pyrrolopyrimidinone I, the pyrrolopyrimidinone II, the pyrrolopyrimidinone III and other LncRNA BANCR can be used for developing and preparing a medicament for inhibiting ovarian cancer liver metastasis.
The foregoing embodiments are provided to illustrate the present invention more fully, but those skilled in the art will appreciate that the scope of the present invention should not be limited to the specific embodiments described above.
Claims (1)
1. Use of an inhibitor of LncRNABANCR in the manufacture of a medicament for inhibiting liver metastasis from ovarian cancer, wherein the inhibitor of LncRNABANCR is selected from the group consisting of compounds having the following structures:
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